Legumain-Cleavable Top1i Antibody-Drug Conjugates without Self-Immolative Spacers Demonstrate Potent Antitumor Activity

Abstract

Virtually all antibody-drug conjugates employ a cleavable linker that is attached to a self-immolative spacer element. The linker and self-immolative spacer are widely known to have a dramatic influence on ADC stability, pharmacokinetics, and therapeutic efficacy. In 2021, our group described a highly polar legumain-cleavable linker that could be used to generate highly potent MMAE-based ADCs. Herein, we build on this finding by describing the design of legumain-cleavable ADCs that release a potent topoisomerase-I inhibitor (TOP1i) without the need for a self-immolative spacer. These ADCs employ an α-TROP2 antibody to target various pancreatic cancer lines. We directly compare their potency, stability, efficacy, and pharmacokinetics to an industry-standard deruxtecan comparator. We show that our TOP1i ADCs exhibit robust cytotoxicity against various cell lines, exert bystander activity, and elicit exquisite efficacy. We believe that this novel linker technology is now poised to be incorporated into next-generation ADCs for a variety of applications.

Graphical Abstract

INTRODUCTION

The clinical success of antibody-drug conjugates (ADCs) represents a paradigm shift in oncology, as they offer a targeted approach to chemotherapy with reduced adverse effects.¹ ADCs are composed of three key elements: a monoclonal antibody that ensures tumor specificity, a payload that drives cytotoxicity, and a metastable linker that is cleaved upon tumor uptake but maintains stability in circulation.^2,3 The selection of an optimal linker is critical to ADC performance, influencing both efficacy and safety.^2,3 The majority of ADCs additionally incorporate a self-immolative moiety that connects the cleavage element to the payload. This self-immolative spacer, such as para-amino benzyl carbamate (PABC), adds hydrophobicity and mechanistic complexity to the ADC that may complicate therapeutic designs. In 2021, our team introduced a new legumain-cleavable linker, AsnAsn, which takes advantage of this tumor-associated protease to release amine-containing payloads.^4,5 Herein, we introduce a new design of exatecan-based ADCs that are cleaved by lysosomal legumain without the need for a self-immolative spacer.

There are currently 14 ADCs approved by the FDA targeting 8 different tumor-associated antigens. One target of particular interest over the past 10 years is tumor-associated calcium signal transducer 2 (also known as TROP2). TROP2 is a cell-surface protein commonly overexpressed in many epithelial cancers, including colorectal, renal, lung, and breast cancers.⁶ It has been the target of multiple ADCs – one of the most successful being sacituzumab govitecan, which combines an α-TROP2 antibody (sacituzumab) with SN-38, a potent Topoisomerase I inhibitor and the active metabolite of irinotecan. In clinical trials, sacituzumab govitecan demonstrated significant improvement in progression-free and overall survival in patients with triple negative breast cancer (TNBC) as compared to traditional chemotherapies.⁷ A number of follow-on ADCs also targeting TROP2 are in the development pipeline, including datopotamab deruxtecan, which was approved by the FDA during the drafting of this manuscript.

Both of the clinically approved TROP2-targeting ADCs on the market employ a potent topoisomerase I inhibitor (Top1i) as the pharmacologically active payload. Topoisomerase inhibitors exert their cytotoxic effects by stabilizing the Topo-1-DNA cleavage complex, thereby preventing DNA religation and leading to the accumulation of DNA damage, ultimately resulting in apoptosis.⁸ These payloads have also been applied to new generations of ADCs that target antigens beyond TROP2. There has been particular excitement in the ADC field around trastuzumab deruxtecan (Enhertu), which is designed to target HER2-positive breast cancer and features a novel cathepsin L (CatL) cleavable linker connected to an potent Top1i. This ADC has shown exquisite clinical efficacy in HER2-positive breast and gastric tumors, including those with low HER2 expression.⁹ Trastuzumab deruxtecan employs a payload known as DX-8951, which is a derivative of exatecan belonging to the camptothecin family. Unlike earlier Topo-1 inhibitors such as irinotecan and SN-38, exatecan exhibits enhanced solubility, improved stability, and an amine handle for linker attachment, making it a superior choice for ADC development.^10–12 Interestingly, both exatecan and deruxtecan-based ADCs enhance antitumor immunity by increasing tumor-infiltrating dendritic cells and CD8+ T cells, upregulating MHC class I expression on tumor cells, and synergizing with anti-PD-1 therapy.¹³

One of the key features of exatecan is its ability to circumvent multidrug resistance (MDR) mechanisms commonly seen in chemotherapy.^11,14 Studies have shown that exatecan retains its potency in tumors that have developed resistance to other chemotherapeutic agents such as topotecan, 9-aminocamptothecin, and SN-38, each of which is known to be a P-glycoprotein substrate.^11,12,15,16 Additionally, exatecan-based ADCs have demonstrated increased efficacy in preclinical studies compared to SN-38-based ADCs, supporting their continued exploration in clinical settings.^10,12

Both DX-8951 and SN-38 lack the requisite amine functional group that is employed by the vast majority of modern-generation ADCs. Rather, both payloads are connected via an alcohol moiety via a self-immolative carbonate or hemiaminal spacer. This spacer element (in the case of SN-38) creates stability issues that have resulted in premature payload release.^17,18 In order to overcome this, and also to take advantage of its increased potency, many teams have focused their efforts on the closely related exatecan core, which contains a primary amine suitable for linker attachment. However, conjugation of exatecan using traditional linker technology, such as mcValCitPABC, has resulted in ADCs with unacceptably high aggregation and poor loading.^10,19 As shown in Table 1, attachment of exatecan to α-HER2 (trastuzumab) via the mcValCitPABC linker results in ~ 20% aggregation. This has prompted the development of novel linker technologies that results in complex ADC designs that incorporate hydrophilic polymers appended to the linker (Figure 1). For example, Schmitt reports trastuzumab-exatecan conjugates that employ a PEG-containing phosphonamidite conjugation moiety that results in low aggregation rates and high loading.¹⁰ Similarly, Weng introduced a polysarcosine-containing PABC spacer that enabled the preparation of high loaded ADCs with excellent solubility.¹² The addition of these polymeric elements into the ADC design demonstrates the need for careful linker design for the delivery of exatecan-based payloads.

Table 1.

Characterization and Cytotoxicity of HER2 Legumain-Cleavable Top1i ADCs^a

ADC	DAR	% Agg.	HIC rt shift from naked mAb (min)	SKBR3 IC50 (μg/mL)
α-HER2_mcValCitPABC_Exatecan	7	19.3	+1.95	0.035 ± 0.006
α-HER2_mcGlyPheGlyGly_DX-8951	8	<5	+2.46	0.074 ± 0.01
α-HER2_mpAsn_Exatecan	7.6	<5	+0.43	>3
α-HER2_mpAsnAsn_Exatecan	6.2	<5	+0.40	0.035 ± 0.008
α-HER2_mpGlyAsnAsnGly_Exatecan	8	<5	+0.69	0.033 ± 0.004
α-HER2_mpGlyAsnAsn(β-Ala)_Exatecan	8	5.8	+0.70	0.043 ± 0.02
α-HER2_mpGlyAsnAsn(GABA)_Exatecan	8	<5	+0.89	0.042 ± 0.006
α-HER2_mpGlyAsnAsnGlyGly_Exatecan	7.8	<5	+0.74	0.046 ± 0.006
α-HER2_mpAsnAsnPABC_Exatecan	7.8	<5	+1.56	0.060 ± 0.01

^aThe table details the drug-antibody ratio (DAR), the percent aggregation (%Agg), HIC retention time shifted from monoclonal antibody (mAb), and IC₅₀ values of ADCs against HER2-expressing SKBR3 cells. Data showed that legumain-cleavable Top1i ADCs exhibited less aggregation and were less hydrophobic compared to ADCs employing traditional linkers (ValCitPABC). All AsnAsn-based ADCs exhibited equivalent or improved cytotoxicity to the industry-standard comparators.

Figure 1.

Schematic diagram showing the challenge of conjugating exatecan using traditional linker technology, previously reported solutions,^10,12 and our proposed solution.

Both of the aforementioned approaches for conjugation of exatecan rely on cathepsin-B cleavable linkers (ValAla or ValCit) and a self-immolative PABC spacer element. Both of these elements add hydrophobicity to the overall conjugate that must be counterbalanced by the hydrophilic polymer. In 2021, our team introduced a new legumain-cleavable linker, AsnAsn, which takes advantage of this tumor-associated protease to release amine-containing payloads in the absence of a self-immolative spacer.^4,5 As demonstrated by the present work and our prior publications, the combination of the hydrophilic AsnAsn peptide linker and the removal of the PABC-spacer element has resulted in ADCs that are highly resistant to aggregation and have very favorable properties for development without the need for the addition of complex hydrophilic polymers.

Legumain is a lysosomal cysteine protease that is particularly highly expressed in solid tumors.^20,21 Our previous work demonstrates that these Asn-containing linkers are rapidly cleaved by lysosomal legumain while maintaining excellent stability in plasma.^4,5 Compared to ValCit-PABC linkers, legumain-cleavable linkers exhibit improved stability against neutrophil elastase, potentially reducing adverse effects such as neutropenia.²² ADCs incorporating these new linkers demonstrate potent and selective cytotoxicity, comparable to traditional ValCit ADCs.⁴ Furthermore, legumain-cleavable ADCs show similar or enhanced cytotoxicity against various tumors, even those with low legumain expression.⁵ These findings suggest that Asn-containing linkers have significant potential in ADC development. Indeed, a legumain-cleavable ADC from Vincerx Pharma targeting CD123 has advanced into a Phase 1 clinical trial (NCT06034275) for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). This ADC combines a CD123 antibody with a potent kinesin spindle protein inhibitor (KSPi) payload, linked via a legumain cleavable linker.²³ Other teams have disclosed Asn-containing ADC designs in the patent literature (see WO2022048883, WO2023173121, and WO2024015229), but to our knowledge only our design and the Vincerx designs have been described in the peer-reviewed literature.

Herein, we build on our prior studies and now report our efforts to design legumain-cleavable Top1i ADC technology. Our ADCs differentiate themselves from prior exatecan technology through the integration of legumain-cleavable linkers which do not require the incorporation of hydrophobic self-immolative spacers. We demonstrate that these ADCs release exatecan derivatives that, while less potent than exatecan itself, maintain exquisite efficacy and stability in a variety of preclinical assays. Moreover, we show that a subset of these ADCs maintain bystander activity that is important for the treatment of tumors with heterogeneous antigen expression. Finally, we show that these ADCs have excellent stability in plasma and improved ADC exposure as compared to deruxtecan technology. Having validated this technology using α-TROP2 and α-HER2 vehicles, we believe that this technology is now poised for application in a variety of other tumor-targeting delivery systems.

RESULTS AND DISCUSSION

In our prior work, we performed an “enzyme agnostic” screen of 75 peptide FRET pairs, demonstrating that Asn-containing peptides exhibited rapid and specific cleavage by lysosomal legumain while retaining robust stability in human and mouse plasma. Building on this finding, we designed monomethyl auristatin E (MMAE) ADCs incorporating the legumain-cleavable linker and went on to show that they exhibited potent cytotoxic activity and were not cleaved by neutrophil elastase.⁴ Finally, we also demonstrated that legumain-cleavable MMAE ADCs elicit potent activity in both high and low legumain-expressing cell lines and exhibit comparable efficacy in xenograft studies to the conventional vcMMAE conjugates.⁵ Having identified novel Asn-containing ADC linkers, we endeavored to apply this technology to the development of exatecan-based ADCs.

Validation Legumain-Cleavable Top1i Technology Using α-HER2 ADCs.

As an initial effort, we attached both Asn and AsnAsn to the exocyclic amine of exatecan, resulting in mpAsn_Exatecan (4) and mpAsnAsn_Exatecan (5). (Scheme 1) Importantly, trityl protecting groups were used to prevent dehydration of the primary amide during the coupling process. The resulting linker-payloads were attached to a model α-HER2 antibody (trastuzumab) using endogenous cysteine chemistry. The process of conjugation to the endogenous cysteine residues was initiated by the reduction of the antibody with tris(2-carboxyethyl)phosphine (TCEP) to cleave the interchain disulfide bonds, thereby exposing 8 sites for conjugation. This was immediately followed by conjugation of the linker-payload to exposed thiols, resulting in ADCs α-HER2_mpAsn_Exa and α-HER2_mpAsnAsn_Exa with a drug-to-antibody ratio (DAR) of 7.6 and 6.2, respectively. (Table 1) As a control ADC, we also prepared α-HER2_ValCitPABC_Exa. These three ADCs were incubated with rat lysosomal extracts while using LCMS/MS to monitor the release of free exatecan. As anticipated, the ValCitPABC linker was rapidly cleaved, resulting in nearly complete release of exatecan within 4h. (Figure S1) In contrast, no exatecan release was observed from the Asn_exa and AsnAsn_exa ADCs.

Scheme 1. Preparation of Linker Payloads^a.

^aReagents: (a) mc-OSu/DIPEA; (b) Fmoc-Asn(Trit)–OH/PYBOP/DIPEA; (c) piperidine; (d) mp-OSu/DIPEA; (e) TFA/Et₃SiH; (f) HATU/DIPEA/Fmoc-Asn(Trit)–OH followed by piperidine; (g) CO(PNP)2/DIPEA; (h) Exatecan/2,6-lutidine/HOAt/DIPEA; (i) PYBOP/Exatecan/DIPEA.

The lack of exatecan release from the Asn-linked exatecan ADCs suggested that the exocyclic amide on the F ring of exatecan is sterically prevented from engaging with lysosomal legumain. In order to move the cleavage site away from the F ring, we envisioned the insertion of a spacer element between the F ring and the Asn cleavage point. (Figure 1) With this in mind, we prepared a series of exatecan-based linker-payloads in which a short spacer element was introduced adjacent to the cleavage site. As an initial proof of concept, we prepared mpAsnAsnPABC_Exatecan (8) via a 6-step sequence beginning from Fmoc-Asn(Trit)–OH and PABA, as shown in Scheme 1. The intermediate (7) was coupled with a preactivated maleimide-propionyl ester, followed by activation of the benzylic alcohol with PNP-carbonate. Addition of exatecan followed by deprotection of the trityl groups provided the final product in ~ 31% overall yield.

Additional spacer elements of varying length were incorporated between the Asn cleavage site and the F-ring amino substituent. As shown in Scheme 1, various AsnAsn-containing peptides (9a-9e, Genscript) were coupled directly to exatecan to provide 10a–10e generally in high yield. In some cases, minor amounts of an epimer (presumably epimerization of the C-20 hydroxyl) were observed – but could be removed during the HPLC purification step. This epimerization presumably arose due to the highly acidic conditions used for the removal of the trityl protecting group, and was generally mitigated by keeping reaction times short and avoiding elevated temperatures. All linker-payloads were stored as DMA stock solution at −80 °C until the time of conjugation.

The above linker payloads were once again conjugated to trastuzumab (α-HER2), resulting in the conjugates shown in Table 1. Note that we included the well-studied Enhertu linker payload (GGFG-DX-8951) and ValCitPABC_Exatecan as comparators. The resulting AsnAsn-linked ADCs exhibited DAR values within the range of 6–8. As anticipated, the conventional ValCitPABC-Exa ADC had a very high level of aggregation (19%) and a significant shift in hydrophobic interaction chromatography (HIC) retention time as compared to the naked α-HER2 antibody (+1.95 min). This indicates that the hydrophobic ValCitPABC linker is introducing problematic biophysical properties to the ADC, which may greatly complicate development efforts. The Enhertu comparator (GGFG-DX-8951) also showed a high HIC shift (+2.46 min). Remarkably, almost all ADCs with the AsnAsn linker had low aggregation (<5%) and a low HIC shift (~0.7 min), indicating that the polar AsnAsn linker offsets the hydrophobicity of the exatecan. Interestingly, the addition of a PABC self-immolative spacer (α-HER2_mpAsnAsnPABC_Exa) partially negated these effects, showing a much higher HIC shift (+1.56 min), thus illustrating the negative impact of the commonly used PABC element on ADC hydrophobicity.

The cytotoxicity of the α-HER2 ADCs was evaluated against a well-studied HER2+ breast cancer cell line, SKBR3. As shown in Table 1, the ADC with a single Asn attached to the exatecan was very weakly active against SKBR (IC50 > 3 μg/mL). This result is consistent with the lysosomal cleavage study mentioned above (Figure S1), which demonstrated no cleavage of the Asn-Exa bond. Interestingly, α-HER2_mpAs-nAsn-Exa exhibited potent cytotoxicity against SKBR3 (0.035 μg/mL) despite the fact that this ADC was shown to release little or no exatecan during lysosomal incubation (Figure S1). This suggests that the active released species (presumably Asn-Exa) is an effective Top1 inhibitor. Introduction of the spacer element (Gly, GlyGly, βAla, GABA) resulted in ADCs with excellent potency against SKBR3 (IC₅₀ 0.033–0.046 μg/mL). In summary, all of the α-HER2 AsnAsn-linked ADCs exhibited comparable or improved cytotoxicity and biophysical properties to the ValCitPABC-Exa and GGFG-DX-8951 conjugates, thus validating our ADC design shown in Figure 1.

Preparation and Characterization of Legumain-Cleavable α-TROP2 ADCs.

While the preliminary utility of this technology was demonstrated using an α-HER2 platform, our long-term goal is to develop ADCs for the treatment of nonsmall cell lung and pancreatic cancer. TROP2 is a cell-surface protein commonly overexpressed in many epithelial cancers, including colorectal, renal, lung, breast, and pancreatic cancers.^6,24 TROP2 overexpression in multiple tumor types has made this a useful biomarker for various cancers. TROP2 has been suggested to be a particularly promising target for pancreatic cancer because of its overexpression (both mRNA and protein) in patient samples.²⁴ TROP2 expression is associated with tumor growth, invasion, and metastasis, and its overexpression correlates with poor clinical outcomes in cancers such as prostate and breast.^25,26 TROP2-targeted antibody-drug conjugates (ADCs) have shown promising efficacy in treating diverse solid tumors, particularly breast cancer and urothelial carcinoma.²⁷ Sacituzumab govitecan, a TROP2-targeted ADC, has been approved for treating metastatic triple-negative breast cancer and hormone receptor-positive, HER2-negative breast cancer.^25,28

Similar to the trastuzumab conjugates described above, conjugation was conducted by reducing the interchain disulfide bonds of the monoclonal antibody (Sacituzumab), thereby exposing multiple thiols for conjugation. Table 2 shows the list of α-TROP2 ADCs that were prepared from the various legumain cleavable linker-payloads. ADCs using industry standard technology (mcGlyGlyPheGly_DX-8951 and mcValCitPABC_Exa) were also prepared. Most ADCs exhibited DAR values between 6 and 8. Consistent with our preparation of α-HER2 conjugates, the mcValCitPABC-Exa ADC exhibited high aggregation and HIC shift (7% and +2.9 min, respectively) while the GGFG-DX-8951 ADC exhibited low aggregation but a significant HIC shift (+1.5 min). In contrast, the AsnAsn ADCs with a spacer element all showed low aggregation (<5%) and low HIC shift (+0.2–0.3 min), once again illustrating the potential biophysical advantages of this new linker system. Note that slight increases in hydrophobicity were observed with longer spacer elements, with HIC shift gradually increasing from 0.23 to 0.33 min as the spacer increased in length from 1 carbon (Gly-Exa) to 5 carbons (AHX-Exa). The impact of the reduced hydrophobicity of these ADCs was further demonstrated by a forced degradation study (Figure S2) showing that the ValCitPABC ADC rapidly aggregated at 45 °C while the legumain cleavable ADCs were highly resistant to aggregation, retaining low levels of aggregation for at least 1 week at this elevated temperature. Notably, the rate of aggregation was dependent upon the antibody itself – with ADCs derived from the α-HER2 mAb (trastuzumab) showing much higher resistance to aggregation than the ADCs derived from the α-TROP2 mAb (sacituzumab).

Table 2.

Characterization and Cytotoxicity of α-TROP2 Legumain-Cleavable Top1i ADCs^a

ADC	DAR	% Agg	ΔHIC retention time (DAR8)	BXPC-3 (IC50, μg/mL) (Trop2+)	CFPAC-1(IC50, μg/mL) (Trop2+)	AsPC-1(IC50, μg/mL) (Trop2 null)
α-TROP2_mcValCitPABC_Exa	7.6	7	+2.9	0.07 ± 0.009	0.21 ± 0.04	6.1 ± 1.8
α-TROP2_mcGlyGlyPheGly_DX-8951	7.9	<5	+1.5	0.06 ± 0.007	0.27 ± 0.08	>30
α-TROP2_mc_Exa	8.0	<5	+2.0	6.2 ± 1.6	>30	>30
α-TROP2_mpAsn_Exa	7.2	<5	+0.21	2.1 ± 0.6	>30	>30
α-TROP2_mpAsnAsn_Exa	5.6	<5	+0.15	0.12 ± 0.01	0.20 ± 0.06	>30
α-TROP2_mpGlyAsnAsnGly_Exa	7.1	<5	+0.23	0.04 ± 0.004	0.05 ± 0.01	>30
α-TROP2_mpGlyAsnAsn(β-ala)_Exa	7.0	<5	+0.25	0.01 ± 0.002	0.10 ± 0.03	>30
α-TROP2_mpGlyAsnAsn(GABA)_Exa	6.5	<5	+0.29	0.07 ± 0.009	0.06 ± 0.01	>30
α-TROP2_mpGlyAsnAsn(AHX)_Exa	4.5	<5	+0.33	0.18 ± 0.02	0.52 ± 0.08	>30
α-TROP2_mpGlyAsnAsnGlyGly_Exa	6.3	<5	0.28	0.04 ± 0.005-	0.04 ± 0.009	>30
IgG_mpGlyAsnAsnGly_Exa	8.0	<5			19 ± 54
IgG_mpGlyAsnAsn(GABA)_Exa	7.9	<5			8.5 ± 2.9
IgG_mpGlyGlyPheGly_DX-8951	8.5	<5			>30

^aData demonstrating the drug-antibody ratio (DAR), the percent aggregation(%Agg), and Δ HIC retention time (DAR8) of α-TROP2 ADCs. The table also provides the IC₅₀ values of ADCs against high TROP2-expressing cell lines(BxPC-3 and CFPAC-1) and a low TROP2-expressing cell line(AsPC-1). This data shows that legumain-cleavable Top1i ADCs exhibit reduced aggregation and hydrophobicity compared to ADCs employing traditional linkers (ValCitPABC and GGFG) while maintaining potency against various TROP2-expressing cell lines.

Overall, the characterization of these ADCs shows that our novel legumain-cleavable Top1i ADCs are more hydrophilic and less prone to aggregation than industry-standard technology. This is particularly important because aggregation and hydrophobicity are important predictors of systemic clearance and off-target toxicity.^29–31

Cytotoxicity of α-TROP2 ADCs.

The α-TROP2 ADCs were evaluated against two TROP2+ cell lines (BxPC-3 and CFPAC-1) and one TROP2 null cell line (AsPC-1). BxPC-3 is a pancreatic epithelial tumor cell line that has been shown to have high TROP2 expression.³² The CFPAC-1 cell line, derived from a cystic fibrosis patient with pancreatic adenocarcinoma, displays epithelial morphology and expresses cytokeratin and oncofetal antigens characteristic of pancreatic duct cells.³³ In pancreatic cancer, TROP2 expression levels vary among cell lines, with BxPC-3 showing high expression (nTPM = 1217), CFPAC-1 having moderate expression (nTPM = 620), and AsPC-1 exhibiting low levels (nTPM = 0.2).³⁴ Significantly, BxPC-3 (TROP2 high) lacks a KRAS mutation that is commonly found in pancreatic cancer, while CFPAC-1 (TROP2 moderate) and AsPC-1 (TROP2 low) both contain KRAS mutations.

As shown in Table 2, ADCs with noncleavable linkers (α-TROP2_mc_Exatecan and α-TROP2_mpAsn_Exatecan) exhibit minimal cytotoxicity against all three cell lines. This is consistent with the weak activity of the aforementioned α-HER2 mpAsn ADC. Incorporation of the cleavable AsnAsn linkers resulted in antigen-dependent cytotoxicity as evidenced by the high potency against TROP2-expressing cells (BxPC3 and CFPAC-1) and low potency against TROP2-null cells (AsPC-1). Consistent with the antigen-dependent cytotoxicity, it was also observed that the isotype control ADCs elicited 150- 400x reduced activity against TROP2-expressing CFPAC1 cells (as compared to the matched α-TROP2 ADCs). Interestingly, we noted that the addition of the spacer element between the Exa and the AsnAsn increased the activity of the ADCs. This is illustrated by comparing the α-TROP2_mpAsnAsn-Exa (0.12–0.20 μg/mL) to the α-TROP2_mpGlyAsnAsnGly_Exa (0.04–0.05 μg/mL). However, as the spacer length increases, the activity seems to decrease, culminating in the rather low potency of the AHX-derived ADCs (IC₅₀ 0.2–0.5 μg/mL). ADCs with a short spacer element (Gly and βAla) exhibited 2–5x higher potency than the industry standard comparators (GGFG-DX-8951 and ValCitPABC-Exa). Taken together, these results show that introduction of a short spacer element between the AsnAsn cleavage motif and the exatecan payload results in ADCs that have excellent potency and target specificity – without the need for a self-immolative spacer (such as PABC) that introduces additional complexity and hydrophobicity.

Lysosomal Catabolism and Payload Release.

Having established the potency of the legumain-cleavable Top1i ADCs in various cell lines, we next undertook lysosomal catabolism experiments designed to elucidate the mechanism of action – with the goal of unambiguously identifying the released species responsible for Top1 inhibition. With this goal in mind, α-TROP2_mpGlyAsnAsnGly_Exa, α-TROP2_mpGlyAsnAsn-βAla_Exa, and α-TROP2_mpGlyAsnAsnGABA_Exa were treated with rat liver lysosomes, and the media was monitored by LCMS/MS for the release of the three anticipated species shown in Figure 2. Cleavage of the Asn peptide linker resulted in the rapid and complete release of Gly-Exa, βAla-Exa, and GABA-Exa, respectively (Figures 2A–2C). Importantly, only trace amounts of free exatecan were observed (<1% of total free payload), strongly suggesting that the pharmacology of these ADCs is not driven by exatecan, but rather by the F-ring modified species. Pretreatment of the lysosomes with a selective legumain inhibitor completely blocked payload release from the three AsnAsn-linked ADCs, strongly supporting the involvement of this enzyme in the catabolism. In contrast, catabolism of α-TROP2_mcValCitPABC_Exa (Figure 2D) and α-TROP2_GlyGlyPheGly_Dx8951 (Figure 2E) were not impacted by RR11a. These results provide clear evidence of the identity of the resulting released species for the AsnAsn-linked ADCs and the catabolic dependence on the legumain enzyme.

Figure 2.

Lysosomal catabolism studies of the three lead ADCs and two comparators demonstrated the dependence on legumain for payload release. (A–E) Rapid and complete payload release was observed during a 30 min incubation with rat liver lysosomes. Only trace amounts of free exatecan were released from the 3 AsnAsn-linked ADCs. (F) Pretreatment with a legumain inhibitor (RR-11a) blocked payload release from the AsnAsn linked ADCs but not from the ValCitPABC or GGFG-linked ADCs.

Cytotoxicity of the Released Payload.

ADCs are generally designed to employ payloads with exceptional potency, typically in the low nanomolar or picomolar range. The released species from the legumain-cleavable ADCs were prepared by the simple two-step procedure shown in Scheme 2. Note that these molecules have been previously reported in the literature.³⁵ Table 3 illustrates the IC₅₀ values of exatecan and DX-8951 compared with the three released species (Gly_Exatecan, βAla_Exatecan, and GABA_Exatecan). Consistent with literature reports, exatecan was exceptionally potent against all three cell lines, with IC₅₀ values ~ 5–10x lower than DX-8951. Surprisingly, we found that the Gly_Exa derivative was ~ 5x less potent than DX-8951, while the βAla_Exa and GABA_Exa were ~ 20x-150x less potent. This was unexpected because the corresponding ADCs (Tables 1 and 2) were equipotent or even more potent than the GGFG-DX-8951 ADCs. The potency of the ADCs combined with the weak activity of the released payload is suggestive of poor permeability. Indeed, it is widely known that the ADC delivery mechanism is not reliant on passive permeability, thereby allowing the effective use of many poorly permeable pay-loads.³⁶ One prominent example of this phenomenon is the well-studied MMAF payload, which has very weak cytotoxicity in spite of its use in the generation of high-potency ADCs. Its lack of potency as a free-payload is widely attributed to its poor permeability.³⁷ Consistent with our hypothesis of reduced permeability, we show that the DX-8951 and Gly_Exa ADCs exhibit better bystander activity than the βAla_Exa and GABA_Exa ADCs (vide infra).

Scheme 2. Preparation of Released Species^a.

^aReagents: (a) HATU/DIPEA; (b) TFA/DCM.

Table 3.

Payload Cytotoxicity Assessment^a

payload	BXPC-3 (IC50, nM) (Trop2+)	CFPAC-1 (IC50, nM) (Trop2+)	AsPC-1 (IC50, nM) (Trop2 null)	logP
DX-8951	3.45 ± 0.67	3.26 ± 0.05	18 ± 5.6	0.62
Exatecan	0.79 ± 0.14	0.08 ± 0.02	0.33 ± 0.07	1.39
Gly_Exatecan (12a)	24.5 ± 5.5	11.8 ± 1.77	69.3 ± 35.9	0.24
Beta-Ala_Exatecan (12b)	522 ± 189	244 ± 181	300 ± 117	0.54
GABA_Exatecan (12c)	426 ± 223	47.6 ± 27.7	264 ± 74	0.82

^aIC₅₀ and logP values of exatecan and its derivatives against high TROP2-expressing cell lines (BxPC-3, CFPAC-1) and a low TROP2-expressing cell line (AsPC-1).

Legumain-Cleavable Top1i ADCs Induce Bystander Killing.

ADC bystander killing is an important phenomenon whereby ADCs, after being internalized by antigen-expressing cells, release the payload into the tumor microenvironment, where it diffuses into adjacent cells that may or may not express the target antigen.³⁸ For example, ADCs employing the highly permeable microtubule inhibitor monomethyl auristatin E (MMAE) are well-known to kill both antigen-positive and antigen-null cells in coculture assays. In contrast, MMAF is 50–200x less potent than MMAE due to poor permeability, and therefore does not generally cause bystander killing in coculture assays.^38–40 Bystander activity is often considered a crucial driver of efficacy in solid tumor applications, where antigen heterogeneity may play a significant role. With this understanding, we conducted a coculture of TROP2-positive cells (BxPC-3) and TROP2-null cells (AsPC-1). In order to facilitate independent monitoring of the two cell types, the BxPC3 cells were transfected to express green fluorescent protein (GFP) while the ASPC1 cells were transfected to express red fluorescent protein (RFP). Cells were cultured in a 4:1 ratio (BxPC3:ASPC1) for 5 days in the presence or absence of ADC and were monitored by live cell imaging. After incubation, cells were harvested, stained with Cy5-labeled α-TROP2, and assessed using flow cytometry. Figure 3 shows the end point images of ADC treatments at various concentrations and Figure 4 shows cell counts of both TROP2-positive (BxPC3) and TROP2-null (AsPC-1) cells. Results showed that MMAF ADC (negative control) showed no evidence of bystander killing as observed by the cytotoxicity against BxPC-3 and not AsPC-1 (Figure 4d). In contrast, the MMAE ADC and GGFG-Dxd ADC (positive controls) demonstrated bystander killing as evidenced by the elimination of the AsPC-1 (TROP2-null) cells (Figure 4e/f). These results are visually illustrated by comparing the lack of RFP-positive (AsPC-1) cells in Figure 3b/c as compared to Figure 3a. Turning to the legumain-cleavable ADCs, we found that α-TROP2_GlyAsnAsnGly_Exa effectively killed both antigen-positive BxPC3 and antigen-null ASPC1 at all three concentrations tested (3, 10, and 30 μg/mL) (Figure 3a/4a). However, the α-TROP2_GlyAsnAsn(βAla)_Exa and α-TROP2_GlyAsnAsn(GABA)_Exa ADCs only killed TROP2-null ASPC1 cells at the highest concentrations (Figure-. 3a/b, 4a/b). This is consistent with the aforementioned data showing that the βAla_Exa and GABA_Exa payloads have weak cytotoxicity, likely due to poor permeability. It is important to note that none of these ADCs significantly kill AsPC-1 cells in the absence of BxPC-3 cells, even at concentrations of 30 μg/mL. (see Table 2) Taken together, this data demonstrates that bystander activity can be “tuned” by changing the length of the spacer element between the Asn-containing linker and the F-ring of the exatecan.

Figure 3.

ADC bystander activity. End point live-cell images of the cocultured GFP-labeled TROP2-expressing cells (BxPC-3) and RFP-labeled TROP2-null cells (AsPC-1) with seeding ratios of 4:1. The indicated ADC (a–f) was added, and cells were incubated for 5 days prior to imaging.

Figure 4.

ADC bystander killing assessment. Flow cytometry analysis of cocultured GFP-labeled TROP2-expressing cells (BxPC-3) and RFP-labeled TROP2-null cells (AsPC-1) with seeding ratios of 4:1. The indicated ADC (a–f) was added, and cells were incubated for 5 days prior to flow cytometry quantification. TROP2_mcMMAF and TROP2_vcMMAE were used as negative and positive controls, respectively. Cells were stained with Cy5-labeled α-TROP2 antibody and were analyzed using a BD Accuri 6 flow cytometer. These data demonstrate that the legumain-cleavable ADCs effectively exert bystander killing.

To further understand this data, BxPC-3 cells (high-TROP2) were incubated with the same set of ADCs for 4 days, and media samples were collected for LCMS/MS quantification of released payload. The results (Figure S3) show that, among the legumain-cleavable ADCs, the Gly_Exatecan ADC exhibits the highest release while the GABA_Exatecan ADC exhibits the lowest release. While the media concentration is somewhat below the IC₅₀ of the released payloads (Table 3), it is important to note that the local concentration in the microenvironment surrounding the BxPC-3 cells is likely to be far higher than the concentration in bulk solution. Importantly, the isotype control ADCs did not exhibit any measurable media levels of payload, strongly suggestive that the payload arises from TROP2-mediated uptake followed by diffusion-mediated release. In contrast, while α-TROP2_GGFG-DX8951 exhibited much higher levels of payload release, its matched isotype control also showed very high levels of payload release, suggestive of linker instability, perhaps mediated by extracellular CatL. This is consistent with a recent report showing that CatL drives activity of Enhertu even in antigen-null cells.⁴¹

Legumain-Cleavable Top1i ADCs Induce DNA Damage.

To confirm the mechanism of action of the ADCs, we performed a series of experiments evaluating markers of apoptosis (caspase 3 and PARP1) and DNA damage (H2AX). TROP2-expressing cells (BxPC-3) were treated with TROP2-targeting ADCs and their matched isotype comparators at concentrations ranging from 0 to 10 μg/mL. After 72h, cells were fixed, permeabilized, and stained with antibodies specific for the selected markers and analyzed by flow cytometry. (Figure 5) Statistical differences were assessed between the isotype control and TROP2-targeting ADCs. H2A.X is a validated marker for DNA damage (commonly associated with Top1 inhibition), while caspase-3 and PARP-1 are apoptotic markers.^42–44 As anticipated, α-TROP2_GlyAsnAsnGly_Exa, α-TROP2_GlyAsnAsn(GABA)_Exa, and α-TROP2_GlyGly-PheGly_DX-8951 each promoted significant upregulation of H2A.X as compared to matched isotype controls. (Figure 5a–c) Interestingly, the effect was most pronounced at the lowest concentration tested (0.1 μg/mL). This concentration is well above the IC₅₀ of the ADCs (Table 2). At the highest concentration (10 μg/mL), we observed slight increases in H2A.X for the isotype control samples – suggestive of nonspecific uptake. Likewise, all three ADCs (α-TROP2_GlyAsnAsnGly_Exa, α-TROP2_GlyAsnAsn(GABA)_Exa, and α-TROP2_GlyGlyPheGly_DX-8951) showed significant upregulation of Caspase-3 and PARP1 at the lowest concentration (0.1 μg/mL) while the matched isotype control elicited only insignificant upregulation. As the concentration increased, however, the isotype controls also promoted upregulation of Caspase 3 and PARP1 – again suggestive of modest nonspecific uptake at higher concentrations. Taken together, these results suggest that at low concentrations (below 1 μg/mL), the α-TROP2 ADCs (including the legumain cleavable Top1i derivatives) selectively promote DNA damage and apoptosis. However, at higher concentrations, nonspecific uptake drives DNA damage and apoptosis via nonantigen-mediated uptake pathways.

Figure 5.

Upregulation of DNA markers. BxPC-3 Cells were treated with ADCs (TROP2 targeting and their isotypes) and incubated at 37 °C for 72 h before flow cytometry was conducted to assess the upregulation of DNA damage marker(H2A.X) and apoptotic marker (Caspase-3 and PARP1). Panels (a)–(c) show the MFI of H2A.X following treatment with the legumain cleavable ADCs (a, b) or the deruxtecan ADC (c). Panels (d)–(f) show the MFI of PARP1 following treatment with the legumain cleavable ADCs (d, e) or the deruxtecan ADC (f). Panels (g)–(i) show the MFI of caspase-3 following treatment with the legumain cleavable ADCs (g, h) or the deruxtecan ADC (i).

Legumain-Cleavable Top1i ADCs Elicit Potent Efficacy in a CFPAC1 Xenograft Model.

Having characterized ADCs and assessed the efficacy of these ADCs in vitro, we proceeded to assess prioritized ADCs in vivo. A CFPAC1 xenograft efficacy study was conducted by implanting ~ 7.5 million cells subcutaneously into the right flank of 8-week-old male and female nude (Nu/J) mice (Charles River Laboratories). Tumors were allowed to grow until volumes reached 75–150 mm³, at which point a single dose of ADC was administered. Treatment groups included α-TROP2_-GlyAsnAsnGly_Exa, α-TROP2_GlyAsnAsn(GABA)_Exa, α-TROP2_GlyGlyPheGly_DX-8951 at 3 mg/kg and 10 mg/kg, respectively; Isotype controls (IgG_GlyAsnAsnGly_Exa, IgG_GlyAsnAsn(GABA)_Exa) at 10 mg/kg, and naked mAb control (αTROP2 monoclonal antibody) at 10 mg/kg. Eight mice, consisting of 4 males and 4 females, were randomly assigned to each treatment group. Tumor measurements were taken every 3 days, and mice were sacrificed when tumor volume reached ~ 1000 mm³. Notably, all TROP2-targeted ADCs exhibited nearly complete tumor regression, while little or no tumor regression was observed in the isotype control-treated animals or mAb-treated animals. (Figure 6a/c) No weight loss or behavioral abnormalities were noted during the study, suggestive of an acceptable safety profile. (Figure 6b/d)

Figure 6.

Xenograft efficacy study. Nu/J mice (4F/4 M per group) with TROP2+ BxPC3 tumors of ~100 mm³ were treated with α-TROP2 ADCs or their isotype comparators at 3 mg/kg (left panels) or 10 mg/kg (right panels). Both tumor size (a, b) and animal weight (c, d) were assessed over a 30 day period.

Encouraged by these positive results, we proceeded to evaluate the efficacy of the same ADCs in a large tumor model (400–800 mm³) at 3 mg/kg. Once again, all ADCs showed remarkable efficacy (Figure 7). α-TROP2_GlyAsnAsn-Gly_Exa induced dramatic reductions in tumor volume, from an average of ~ 600 mm³ to an average of less than 100 mm³ by day 30. Similar results were seen for α-TROP2_GlyGly-PheGly_DX-8951. Interestingly, α-TROP2_GlyAsnAsn-(GABA)_Exa was not as potent, with tumors initially shrinking to ~ 200 mm³ and then regrowing to 400 mm³ or more by day 30. This is consistent with the aforementioned reduced bystander activity and lower payload potency of this ADC.

Figure 7.

Large tumor xenograft study. Nu/J mice (4M+4F per group) with TROP2+ BxPC3 tumors of ~500 mm³ were treated with α-TROP2 ADCs or their isotype comparators at 3 mg/kg. Tumor size (a) and animal weight (b) were assessed over a 30 day period.

Legumain Cleavable ADCs Are Stable in Mouse and Human Serum.

To assess the plasma stability of ADC,10 μg/mL of α-TROP2_GlyAsnAsnGly_Exa, α-TROP2_GlyAsnAsn(βAla)_Exa, α-TROP2_GlyAsnAsn-(GABA)_Exa, α-TROP2_GlyGlyPheGly_DX-8951, and α-TROP2_mcValCitPABC_Exa were incubated in mouse and human serum for 14 days at 37 °C. Aliquots were collected at various time points and quantified by LCMS/MS. Results showed that TROP2_GlyGlyPheGly_DX-8951 exhibited the lowest stability in both mouse and human serum (Figure 8), suggestive of premature release of payload in circulation. Likewise, the ValCitPABC comparator exhibited some instability in mouse serum, consistent with literature reports of Ces1C-mediated cleavage. In contrast, the legumain-cleavable ADCs exhibited very low levels of payload in mouse serum and no measurable payload release in human serum.

Figure 8.

ADC plasma stability. ADCs were incubated in mouse and human serum at 37 °C for 14 days. Free payload was measured by LCMS/MS.

Legumain-Cleavable Top1i ADCs Exhibit Improved PK Exposure Compared to GGFG-DX8951 ADC.

Finally, the exposure of legumain cleavable ADCs was assessed in mice and rats and compared directly with the deruxtecan (GGFG-Dxd) comparator. Nu/J mice and SD rats were administered a single dose of ADC by IP administration. Total circulating hIgG levels were by ELISA using an anti-hFc detection antibody. Likewise, total ADC levels were assessed using an anti-Dxd detection antibody. (Note that the detection antibody cross-reacted with Dxd and exatecan.) Results are shown in Figures 9 and Tables S1 and S2. The legumain cleavable ADCs exhibited higher exposure (AUC) and lower clearance as compared to the deruxtecan ADC in both rats and mice. The half-life in mice and rats ranged from ~ 100 to ~ 150 h while the clearance ranged from 0.25 to 0.52 mg/h*kg. Encouragingly, both the total exposure and clearance of α-TROP2_GlyAsnAsnGly-Exa were only ~ 30% lower than the exposure and clearance of the sacatuzumab naked antibody. This is particularly remarkable given the high DAR of this ADC (DAR ~ 8) and illustrates the improved biophysical properties imparted by the asparagine-containing linker. In contrast, the matched deruxtecan ADC exhibited a ~ 70% reduction in exposure compared to the naked antibody.

Figure 9.

Pharmacokinetics in mice (a) and rats (b, c). (a) Total hIgG was assessed in Nu/J mice following a 3 mg/kg IP administration. Total hIgG (b) and ADC (c) was assessed in SD rats following a 2.5 mg/kg IP administration.

CONCLUSIONS

Interest in topoisomerase I inhibitor ADCs has dramatically increased over the past five years due to the approval of sacatuzumab govatecan and trastuzumab deruxtecan. In spite of the success of these ADCs, both have exhibited liabilities that result from linker instability and payload hydrophobicity. Herein, we describe a novel approach for addressing these issues that employs a legumain-cleavable AsnAsn linker. Unlike the majority of ADCs linkers, which require a self-immolative spacer element, our linkers are designed to directly release amine-containing payloads without a PABC element. In order to accomplish this, we installed a small spacer element between the exatecan payload and the AsnAsn linker – resulting in ADCs which release an exatecan derivative as the active species.

We show that the resulting ADCs are highly active as α-HER2 or as α-TROP2 bioconjugates, exhibiting ~ 100–1000 fold higher activity than their cognate isotype control ADC. (Table 1, 2) Importantly, the lead ADCs (containing a glycine or βAla spacer) exhibit low levels of aggregation (<5%) and minimal HIC shift, even when conjugated with a DAR of 8. Catabolism studies revealed that the released species (Gly-Exa, βAla-Exa, or GABA-Exa) is rapidly released in a legumain-dependent manner during lysosomal incubation. (Figure 2) Unexpectedly, however, the released species were shown to be less potent than the parent exatecan. (Table 3) In spite of the lower potency, the ADCs exhibit meaningful bystander activity, albeit reduced as compared to deruxtecan. (Figure 3, 4) As anticipated, the ADCs induce markers of DNA damage and apoptosis consistent with the topoisomerase I mechanism of action (Figure 5). Encouragingly, the lead legumain-cleavable ADCs induced rapid tumor regression at doses of 3 mg/kg and 10 mg/kg in BxPC3 tumor-bearing mice. No weight loss or adverse events were observed. The activity of the lead molecule, α-TROP2_GlyAsnAsnGly-Exa, was equivalent or superior to the matched deruxtecan derivative. (Figure 6, 7) Finally, we demonstrated that the legumain-cleavable ADCs have high exposure and low clearance in both mice and rats, with total circulating ADC levels only slightly reduced as compared to the unmodified antibody.

Our ADCs differentiate themselves from prior exatecan technology through the integration of legumain-cleavable linkers, which do not require the incorporation of hydrophobic self-immolative spacers. We demonstrate that these ADCs release exatecan derivatives that, while less potent than exatecan itself, maintain exquisite efficacy and stability in a variety of preclinical assays. Moreover, we show that a subset of these ADCs maintain bystander activity that is important for the treatment of tumors with heterogeneous antigen expression. Finally, we show that these ADCs have excellent stability in plasma and improved ADC exposure as compared to deruxtecan technology. Having validated this technology using α-TROP2 and α-HER2 vehicles, we believe that this technology is now poised for application in a variety of other tumor-targeting delivery systems.

METHODS

General Experimental Information.

All chemical reagents were purchased commercially and were used without further purification. Asn-containing peptides (9a–e) were custom prepared by Genscript. 1H NMR was performed using a Bruker Avance III HD 400 MHz. The identity and purity of all compounds were determined by 1H NMR and LCMS. Antibodies were custom prepared by SydLabs. Cell lines were obtained from ATCC and were cultured under the suppliers’ recommended conditions. All products were >95% purity as determined by HPLC unless otherwise noted.

Ethical statement.

All animal studies followed ACS Ethical Guidelines and were approved and overseen by the Binghamton University Institutional Animal Care and Use Committee (IACUC). Studies were conducted according to IACUC-approved protocol 868–21 and 890–23. This research did not involve human participants.

Linker-Payload Synthesis.

mc_Exatecan (2).

Exatecan mesylate (10 mg, 1 Eq, 19 μmol) was added to a vial containing a solution of N-succinimidyl 6-maleimidohexanoate (12 mg, 2 Eq, 38 μmol) and DIPEA (2.4 mg, 3.3 μL, 1 Eq, 19 μmol) in DMF (0.38 mL). The reaction was stirred for 3 h while being monitored by UPLC. Upon completion, the product was extracted into DCM (10 mL x3) and the resulting organic layers were dried to obtain the crude product, which was purified using preparative HPLC giving 40% isolated yield. LCMS rt = 2.88 min; m/z = 629.4 [M + H]⁺; HPLC Purity = 99.9%; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.08 (s, 1H), 7.78 (d, J = 10.80 Hz, 1H), 7.30 (s, 1H), 6.97 (s, 2H), 6.56–6.40 (m, 1H), 5.55 (dt, J = 4.02 Hz, 8.47 Hz, 1H), 5.41 (s, 2H), 5.23 (d, J = 19.06 Hz, 1H), 5.13 (d, J = 19.06 Hz, 1H), 5.02 (d, J = 20.54 Hz, 1H), 3.16 (t, J = 6.35 Hz, 2H), 3.10 (s, 2H), 2.60 (s, 2H), 2.38 (s, 3H), 2.14 (t, J = 7.20 Hz, 1H), 2.05 (d, J = 10.80 Hz, 2H), 1.85 (dd, J = 1.91 Hz, 7.62 Hz, 2H), 1.61–1.42 (m, 4H), 0.86 (t, J = 7.62 Hz, 3H).

mpAsn_Exatecan (4).

Step 1. Exatecan mesylate (31.5 mg, 1 Eq, 59.3 μmol) and Fmoc-Asn(Trt)–OH (53.0 mg, 1.5 Eq, 88.9 μmol) were dissolved in 2.8 mL dry DMF and treated with PyBOP (46.3 mg, 1.5 Eq, 88.9 μmol) followed by diisopropylethylamine (11.5 mg, 15.4 μL, 1.5 Eq, 88.9 μmol). The reaction was stirred for 4.5 h at room temperature and was monitored by TLC and UPLC. The reaction was quenched with water the crude product was extracted into dichloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to give the crude product which was used without further purification. LCMS rt = 4.32 min; m/z = 1014.5 [M + H]

Step 2: (Compound 3) The product of step 1 was treated with 0.16 mL of DMF followed by piperidine (5.0 mg, 0.04 mL, 1 Eq, 59 μmol) and the mixture was stirred at room temperature for 1.5 h. The reaction was monitored using TLC and UPLC. The product was purified using prep HPLC to give 21 mg of compound 3 that was used immediately in the next step. LCMS rt = 2.86 min; m/z = 792.4 [M + H]

Step 3: The product of step 2 (21 mg) was dissolved in DMF (0.53 mL) and treated with 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanoate (aka ‘mpOSu’) (14 mg, 2.0 Eq, 53 μmol). The reaction was stirred for 17 h and monitored by TLC and UPLC. Upon completion, the solvent was removed the product was purified using silica gel chromatography (EtOAc/Hex). LCMS rt = 3.64 min; m/z = 943.4 [M + H]

Step 4: The product of step 3 was dissolved in 250ul of DCM and treated with a premixed solution of TFA:TES:H₂O (5 mL, 95:2.5:2.5). The reaction was stirred at rt for 60 min then concentrated to dryness and purified using preparative HPLC providing the product in 24% overall yield. LCMS rt = 2.44 min; m/z = 701.3 [M + H]; HPLC Purity = 97%; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.48 (d, J = 8.46 Hz, 1H), 8.17 (d, J = 7.66 Hz, 1H), 7.78 (d, J = 11.01 Hz, 1H), 7.30 (s, 1H), 7.28 (bs, 1H), 6.94 (d, J = 9.89 Hz, 2H), 6.83 (bs, 1H), 5.52–5.46 (m, 1H), 5.41 (d, J = 4.63 Hz, 2H) 5.25 (d, J = 18.98 Hz, 1H), 5.19 (d, J = 18.98 Hz, 1H), 4.46 (q, J = 7.02 Hz, 1H), 3.57–3.48 (m, 3H), 3.18–3.11 (m, 2H), 3.00 (td, J = 3.83 Hz, 2.71 Hz, 1H), 2.92 (s, 1H), 2.42–2.30 (m, 6H), 1.91–1.79 (m, 2H), 1.72 (dt, J = 3.23 Hz, 3.48 Hz, 1H), 0.87 (t, J = 7.24 Hz, 3H).

mpAsnAsn_Exatecan(5).

Step 1. Compound 3 (24.3 mg, 1 Eq, 30.7 μmol) and Fmoc-Asn(Trt)–OH (27.5 mg, 1.5 Eq, 46.0 μmol) was dissolved in 2.8 mL dry DMF and treated with PYBOP (24.0 mg, 1.5 Eq, 46.0 μmol) and diisopropylethylamine (9.92 mg, 13.3 μL, 2.5 Eq, 76.7 μmol). The reaction was stirred for 15 min at room temperature and was monitored by TLC and UPLC. Upon completion, the solvent was removed providing a crude product that was used without further purification. LCMS rt = 3.82 min; m/z = 1370.8 [M + H]

Step 2. The product of step 1 was dissolved in DMF (0.16 mL) and treated with piperidine (0.04 mL, 1 Eq). After 10 min, the solvent was evaporated providing the crude product which was used without further purification. LCMS rt = 3.52 min; m/z = 1148.6 [M + H]

Step 3. The crude product of step 2 (~20 mg) was dissolved in DMF (0.35 mL) and treated with mpOSu (9.3 mg, 2 Eq, 35 μmol). The reaction was stirred at room temperature for 2 h. The solvent was removed and the crude product was used without further purification. LCMS rt = 3.51 min; m/z = 1301.0 [M + H]

Step 4. The material of step 3 was dissolved into 250ul of DCM and treated with 5 mL of TFA:TES:H2O (95:2.5:2.5). After 30 s, the reaction was concentrated to dryness the solid was redissolved in 0.75 mL DMF for purification by preparative HPLC providing the title compound (54% overall yield). LCMS rt = 2.30 min; m/z = 815.4 [M + H]; HPLC Purity = 97%; ¹H NMR (400 MHz, DMSO) ]ISP]δ_H/ppm 8.34–8.11 (m, 3H), 7.73 (dd, J = 5.89 Hz, 11.31 Hz, 1H), 7.29 (d, J = 3.67 Hz, 1H), 7.26 (bs, 1H), 6.96 (s, 2H), 6.90 (s, 1H), 5.42 (s, 2H), 5.28 (d, J = 18.99 Hz, 1H), 5.24–5.14 (m, 2H), 4.53–4.45 (m, 2H), 4.31–4.24 (m, 2H), 3.99 (d, J = 11.79 Hz, 1H), 3.83 (d, J = 11.79 Hz, 1H), 3.46 (d, J = 11.13 Hz, 1H), 3.36 (t, J = 7.60 Hz, 1H), 3.18–3.10 (m, 2H), 2.42–2.34 (m, 6H), 2.33–2.24 (m, 4H), 1.90–1.82 (m, 2H), 1.76 (t, J = 7.86 Hz, 1H), 0.87 (t, J = 7.44 Hz, 3H).

mpAsnAsnPABC_Exatecan (8).

Step 1, NH₂–Asn(Trit)-PAB–OH: Fmoc-Asn(Trt)–OH (1 g, 1 equiv) and PAB–OH (258 mg, 1.25 equiv) were dissolved in 4 mL of DMF giving a dark solution. HATU (764 mg, 1.2 equiv) was added followed by DIPEA (876 μL, 3 equiv) and the reaction was stirred at rt for 1 h. Piperidine (1 mL) was added and the rxn was stirred for 30 min. The product was partitioned between EtOAc/MeOH and 1 M NaOH. The material was dried over MgSO4 and concentrated to give 1.6 g of crude product. The product was purified by silica gel chromatography (0→10% MeOH in DCM) to give 0.57g of crude material that was used directly in the next step (74%). LCMS rt = 2.75 min; m/z = 480.3 [M + H]

Step 2, NH₂–Asn(Trit)Asn(Trit)_PAB–OH (7): NH₂–Asn(Trt)-PAB–OH (478 mg) was dissolved in 4 mL of DMF forming a clear yellow solution which was treated with Fmoc-Asn(Trt)–OH (595 mg, 1 equiv), HATU (455 mg, 1.2 equiv) and DIPEA (521 μL, 3 equiv) was added and the reaction was stirred at rt for 1 h. Piperidine (1.0 mL) was added to the reaction and this was left to stir for 30 min. The product was partitioned between EtOAc/MeOH and 1 M NaOH. The material was dried over MgSO4 and concentrated to give 1.07g of crude product. The crude product was purified by silica gel chromatography (0→10% MeOH in DCM) to give 0.652g of the title compound (78%). LCMS rt = 3.19 min; m/z = 836.4 [M + H]

Step 3, mpAsn(Trit)Asn(Trit)_PAB–OH: A DMF (0.60 mL) solution of compound 7 (25 mg, 1 Eq, 30 μmol) was treated with mpOSu (16 mg, 2 Eq, 60 μmol). After 17h, the reaction was diluted with water and the product was extracted into DCM (3 × 10 mL). The crude product was obtained upon evaporation. LCMS rt = 4.00 min; m/z = 987.6 [M + H]

Step 4, mpAsn(Trit)Asn(Trit)_PAB-OPNP: The crude material from step 3 (20 mg, 0.20 mL of 100 mM, 1 Eq, 20 μmol) was dissolved in DMF (0.20 mL) and treated with diisopropylethylamine (3.9 mg, 5.3 μL, 1.5 Eq, 30 μmol). This mixture was added to a dried vial of bis-PNP carbonate (22 mg, 3.6 Eq, 72 μmol). The vial was flushed with nitrogen and stirred for 12 h and monitored by TLC and UPLC. The crude product was used without purification. LCMS rt = 4.37 min; m/z = 1153.5 [M + H]

Step 5, mpAsn(Trit)Asn(Trit)PABC_Exatecan. The crude reaction from step 4 was directly treated with exatecan mesylate (21 mg, 2 Eq, 40 μmol) followed by 2,6-lutidine (4.3 mg, 4.6 μL, 2 Eq, 40 μmol), 1-hydroxy-7-azabenzotriazole (2.7 mg, 1 Eq, 20 μmol) and diisopropylethylamine (10 mg, 14 μL, 4 Eq, 80 μmol). After 1.5h, the material was purified by preparative HPLC to give the title compound. LCMS rt = 4.25 min; m/z = 1449.7 [M + H].

Step 6, mpAsnAsnPABC_Exatecan (8): A solution of TFA (1.5 mL) and DCM (2.0 mL) was treated with triethyl silane (18 uL, 190 Eq) and added to the product of step 5. The vial was placed in a −20 freezer overnight, and LCMS confirmed the reaction had gone to completion. Upon concentration, the title product was obtained by preparative HPLC purification. LCMS rt = 2.62 min; m/z = 964.5 [M + H]; HPLC Purity = 96%; Overall yield = 31.3%. ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.36 (d, J = 7.34 Hz, 1H), 8.31 (d, J = 7.77 Hz, 1H), 8.06 (d, J = 8.47 Hz, 1H), 7.79 (d, J = 7.77 Hz, 1H), 7.71 (d, J = 8.57 Hz, 2H), 7.48 (bs, 1H), 7.36 (d, J = 8.64 Hz, 2H), 7.32 (s, 1H), 7.03 (bs, 1H), 7.00 (d, J = 3.12 Hz, 2H), 6.89 (bs, 1H), 5.45 (s, 2H), 5.30 (d, J = 16.64 Hz, 3H), 5.09 (bs, 1H), 4.69–4.61 (m, 1H), 4.52–4.43 (m, 1H), 3.62 (t, J = 7.76 Hz, 4H), 3.30–3.20 (m, 1H), 3.18–3.06 (m, 1H), 2.69–2.59 (m, 2H), 2.57 (d, J = 7.10 Hz, 1H), 2.46–2.36 (m, 6H), 2.35–2.36 (m, 1H), 2.24–2.14 (m, 2H), 1.88 (dt, J = 5.98 Hz, 17.39 Hz, 2H), 1.24 (s, 1H), 0.88 (t, J = 7.25 Hz, 3H)

mpGlyAsnAsnGly_Exatecan (10a).

Exatecan mesylate (10 mg, 1 Eq, 19 μmol) and mpGlyAsnAsnGly-OH (9a) (14 mg, 1.5 Eq, 28 μmol) were dissolved in DMF (0.38 mL) and treated with PyBOP (20 mg, 2.0 Eq, 38 μmol) and DIPEA (2.4 mg, 3.3 μL, 1.0 Eq, 19 μmol). The reaction was stirred for 4.5 h at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC giving 20% yield of the isolated product. LCMS rt = 2.32 min; m/z = 929.4 [M + H]; HPLC Purity = 98.2%; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.27–8.15 (m, 4H), 8.07 (d, J = 7.67 Hz, 1H), 7.77 (d, J = 10.93 Hz, 1H), 7.39 (bs, 1H), 7.31 (s, 1H), 7.30 (bs, 1H), 6.97 (s, 2H), 6.94 (bs, 1H), 6.71 (bs, 1H), 6.49 (bs, 1H), 5.58–5.51 (m, 1H), 5.41 (s, 2H), 5.27 (d, J = 18.97 Hz, 1H), 5.18 (d, J = 18.97 Hz, 1H), 4.49–4.42 (m, 1H), 4.40–4.33 (m, 1H), 3.73 (d, J = 6.07 Hz, 2H), 3.62 (dd, J = 2.06 Hz, 3.31 Hz, 2H), 3.57 (t, J = 7.62 Hz, 3H), 3.20–3.12 (m, 2H), 2.54 (t, J = 5.68 Hz, 2H), 2.44–2.33 (m, 6H), 2.24–2.04 (m, 2H), 1.86 (sept, J = 7.86 Hz, 2H), 0.87 (t, J = 7.36 Hz, 3H).

mpGlyAsnAsn(βAla)_Exatecan (10b).

The title compound was prepared in 34% yield following the procedure used for the preparation of compound 10a, except using 9b in place of 9a. LCMS rt = 2.37 min; m/z = 943.8 [M + H]; HPLC Purity = 85.2%; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.49 (d, J = 8.48 Hz, 1H), 8.23 (t, J = 5.65 Hz, 1H), 8.18 (d, J = 7.68 Hz, 1H), 8.12 (d, J = 8.12 Hz, 1H), 7.80 (d, J = 10.87 Hz, 2H), 7.46 (bs, 1H), 7.32 (s, 1H), 7.25 (bs, 1H), 6.99 (s, 3H), 6.81 (bs, 1H), 6.51 (s, 1H), 5.60–5.53 (m, 1H), 5.43 (d, J = 2.75 Hz, 2H), 5.26 (d, J = 18.99 Hz, 1H), 5.18 (d, J = 18.99 Hz, 1H), 4.52–4.41 (m, 2H), 3.68 (d, J = 5.65 Hz, 2H), 3.59 (dd, J = 1.16 Hz, 7.54 Hz, 2H), 3.23–3.14 (m, 2H), 2.68 (quin, J = 1.90 Hz, 1H), 2.46–2.31 (m, 11H), 2.20–2.10 (m, 3H), 1.93–1.81 (m, 2H), 0.88 (t, J = 7.39 Hz, 3H)

mpGlyAsnAsn(GABA)_Exatecan (10c).

The title compound was prepared in 34% yield following the procedure used for the preparation of compound 10a, except using 9c in place of 9a. LCMS rt = 2.41 min; m/z = 957.6 [M + H]; HPLC Purity = 95.2%; Overall yield = 33.9%. ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.40 (d, J = 8.45 Hz, 1H), 8.23 (t, J = 5.81 Hz, 1H), 8.18 (d, J = 7.25 Hz, 1H), 8.09 (d, J = 8.04 Hz, 1H), 7.78 (d, J = 10.92 Hz, 1H), 7.71 (t, J = 5.44 Hz, 1H), 7.45 (bs, 1H), 7.30 (s, 1H), 7.23 (bs, 1H), 6.98 (s, 2H), 6.80 (bs, 1H), 5.74 (s, 1H), 5.59–5.51 (m, 1H), 5.41 (bs, 2H), 5.23 (d, J = 18.86 Hz, 1H), 5.15 (d, J = 18.86 Hz, 1H), 4.50–4.38 (m, 2H), 3.66 (d, J = 5.44 Hz, 3H), 5.58 (t, J = 7.48 Hz, 3H), 3.16 (s, 3H), 3.08–2.99 (m, 2H), 2.56 (d, J = 6.83 Hz, 1H), 2.46–2.35 (m, 6H), 2.20–2.08 (m, 4H), 1.85 (sept, J = 7.77 Hz, 2H), 1.75–1.65 (m, 2H), 0.86 (t, J = 7.39 Hz, 3H)

mpGlyAsnAsn(AHX)_Exatecan (10d).

The title compound was prepared in 17% yield following the procedure used for the preparation of compound 10a, except using 9d in place of 9a. LCMS rt = 2.41 min; m/z = 985.5 [M + H]; HPLC Purity = 98.5%; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.41 (d, J = 8.68 Hz, 1H), 8.24 (t, J = 5.74 Hz, 1H), 8.18 (d, J = 7.49 Hz, 1H), 8.08 (d, J = 7.99 Hz, 1H), 7.78 (d, J = 10.98 Hz, 1H), 7.44 (bs, 1H), 7.30 (s, 1H), 7.24 (bs, 1H), 6.98 (s, 3H), 6.82 (bs, 1H), 5.58–5.51 (m, 1H), 5.41 (s, 2H), 5.22 (d, J = 18.97 Hz, 1H), 5.14 (d, J = 18.97 Hz, 1H), 4.50–4.39 (m, 2H), 3.66 (d, J = 5.49 Hz, 2H), 3.58 (dd, J = 1.99 Hz, 6.24 Hz, 2H), 3.16 (bs, 2H), 2.45–2.35 (m, 6H), 2.18–2.08 (m, 4H), 1.91–1.78 (m, 2H), 1.72 (dt, J = 3.41 Hz, 6.40 Hz, 9H), 1.61–1.51 (m, 2H), 1.41–1.34 (m, 3H), 0.86 (t, J = 7.29 Hz, 3H)

mpGlyAsnAsnGlyGly_Exatecan (10e).

The title compound was prepared in 14% yield following the procedure used for the preparation of compound 10a, except using 9e in place of 9a. LCMS rt = 2.32 min; m/z = 986.8 [M + H]; HPLC Purity = 98.0%; Overall yield = 13.7%. ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.29 (d, J = 8.62 Hz, 1H), 8.22–8.10 (m, 4H), 8.05 (t, J = 5.95 Hz, 1H), 7.78 (d, J = 10.96 Hz, 1H), 7.43 (bs, 1H), 7.30 (s, 1H), 7.23 (bs, 1H), 6.98 (s, 3H), 6.86 (bs, 1H), 5.59–5.53 (m, 1H), 5.42 (s, 2H), 5.25 (d, J = 18.95 Hz, 1H), 5.17 (d, J = 18.95 Hz, 1H), 4.55–4.48 (m, 1H), 4.43–4.37 (m, 1H), 3.71–3.63 (m, 4H), 3.58 (t, J = 7.68 Hz, 4H), 3.20–3.16 (m, 2H), 3.16 (s, 1H), 3.03–2.98 (m, 1H), 2.43–2.35 (m, 7H), 2.24–1.99 (m, 2H), 1.92–1.79 (m, 2H), 0.86 (t, J = 7.52, 3H)

Gly-Exatecan (12a).

Step 1: Exatecan mesylate 1 (25 mg, 1 Eq, 47 μmol) was dissolved in 1 mL dry DMF and treated with diisopropylethylamine (16 μL, 2 Eq, 94 μmol) followed by (tert-butoxycarbonyl)glycine (11a) (12 mg, 1.5 Eq, 71 μmol) and HATU (49 mg, 2 Eq, 94 μmol). The reaction was monitored by UPLC. After 3 h, the reaction was quenched with water and the crude product was extracted into ethyl acetate (20 mL x 3). The organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated to give the crude product that was used without further purification. LCMS rt = 2.97 min; m/z = 593.4 [M + H]⁺.

Step 2: The material from step 1 was dissolved in 1 mL 15% TFA in DCM and stirred at room temperature and monitored using UPLC. After 35 min, the crude mixture was then treated with 15 mL of water and the product was extracted into ethyl acetate (20 mL x 3). The organic extracts dried over anhydrous magnesium sulfate, filtered, and concentrated to give a crude product that was further purified by preparative HPLC, giving 33% isolated yield. LCMS rt = 2.03 min; m/z = 493.4 [M + H]⁺; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.87 (d, J = 8.47 Hz, 1H), 8.10–8.00 (m, 3H), 7.83 (d, J = 10.93 Hz, 1H), 7.33 (s, 1H), 6.53 (bs, 1H), 5.68–5.61 (m, 1H), 5.43 (s, 2H), 5.37 (d, J = 18.85 Hz, 1H), 5.24 (d, J = 18.85 Hz, 1H), 3.56 (bs, 2H), 3.27–3.10 (m, 2H), 2.42 (s, 3H), 2.27–2.21 (m, 1H), 1.90–1.80 (m, 2H), 0.87 (t, J = 7.22 Hz, 3H).

βAla-Exatecan (12b).

Prepared in two steps from exatecan mesylate (1) and Boc-βAla (11b) using the same method as outlined for 12a, giving 63% overall yield. LCMS rt = 2.07 min; m/z = 507.5 [M + H]^{+; 1}H NMR (400 MHz, DMSO) δ_H/ppm 8.66 (d, J = 8.57 Hz, 1H), 7.81 (d,J = 10.96 Hz, 1H), 7.75 (bs, 2H), 7.32 (s, 1H), 6.53 (s, 1H), 5.61–5.54 (m, 1H), 5.42 (s, 2H), 5.25 (dd, J = 7.46 Hz, 18.90 Hz, 2H), 3.22–3.15 (m, 2H), 3.13–2.98 (m, 3H), 2.53 (d, J = 6.83 Hz, 2H), 2.41 (s, 3H), 2.22–2.12 (m, 1H), 1.91–1.80 (m, 2H), 0.87 (t, J = 7.30 Hz, 3H).

GABA-Exatecan (12c).

Prepared in two steps from exatecan mesylate (1) and Boc-GABA (11c) using the same method as outlined for 12a, giving 52% overall yield. LCMS rt = 2.09 min; 521.5 [M + H]⁺; ¹H NMR (400 MHz, DMSO) δ_H/ppm 8.56–8.51 (m, 1H), 7.80 (d,J = 10.95 Hz, 1H), 7.73 (bs, 2H), 7.32 (d,J = 2.17 Hz, 1H), 6.53 (bs, 1H), 5.61–5.54 (m, 1H), 5.42 (s, 2H), 5.20 (q, J = 18.86 Hz, 2H), 3.21–3.14 (m, 2H), 2.89–2.81 (m, 2H), 2.40 (s, 3H), 2.14 (q, J = 5.44 Hz, 2H), 1.94 (d, J = 12.34 Hz, 3H), 1.90–1.81 (m, 3H), 0.87 (t, J = 7.10 Hz, 3H)

Conjugation of Linker Payloads to α-TROP2 (Sacituzumab).

1 mg of Sacituzumab in 5 mM EDTA/PBS was treated with 8eq of 5 mM TCEP and incubated for 2 h at 37 °C. After 2 h of incubation, 12eq of the appropriate exatecan linker-payload was added (as a 5 mM stock in DMA) and left to incubate for 2 h at room temperature. The crude ADC was buffer exchanged into PBS using 10K or 30K concentrator column and resuspended to 1 mL of PBS. ADCs were analyzed by LC-MS, SEC, and HIC to assess DAR, and %aggregation.

Conjugation of Linker Payloads to α-HER2 (Trastuzumab).

Trastuzumab (67 μL of 15 mg/mL stock = 1 mg) was diluted with 417 μL of 5 mM EDTA/PBS. Twelve equivalents of 5 mM TCEP was then added (16 μL of 5 mM solution = 166 μM) and left to incubate at 37 °C for 2 h. After the incubation period, the solution was buffer exchanged into 5 mM EDTA/PBS using 30 kDa spin columns (spun at 15,000g for 5 min) and concentrated to 450 μL. Fifteen equivalents of each linker payload (20 μL of 5 mM stock = 208.5 μM), along with 30 μL DMA (total DMA concentration of 10%) was then added to the solution and the reaction was left to sit at room temperature for 1–2 h. The reaction mixture was then buffer exchanged into 1 mL of PBS using a PD10 (gel filtration) column. ADCs were analyzed by LC-MS, SEC, and HIC to assess DAR, and %aggregation.

Liquid Chromatography Mass Spectrometry.

DAR estimation by of ADCs was performed using LCMS, wherein 15 μL of a ~ 1 mg/mL sample was mixed with 5 μL of 500 mM TCEP, vortexed, and 15 μL was injected into Waters Acquity HPLC equipped with a TUV Detector and Xevo TQD mass spectrometer. The separation was achieved using a Sepax proteomics RP-1000 5 μm column (1000Å and 2.1 × 100 mm) at 80 °C, using a gradient of 5% to 95% B. Solvent A was water with 0.1% formic Acid (FA), and solvent B was acetonitrile with 0.1% FA. The typical injection size was 5–10 μL. The eluent was monitored by UV (220, 260, and 280 nM) and by ESI mass spectroscopy (TQD, ES+). The charge envelope (~700–1700 m/z) was deconvoluted using Maxent1.

Size Exclusion Chromatography.

Aggregation levels were assessed via size exclusion chromatography (SEC) on an Agilent 1260 HPLC with a Tosoh TSKgel G3000 column, injecting 5 μL of 1 mg/mL samples and using a 10% acetonitrile/DPBS buffer. SEC analysis was performed on an Agilent 1260 infinity II with a TUV detector using a TSKGel 3000SW (7.5 mmx30 cm) column. Analysis was performed at room temperature using a 20 min isocratic gradient of phosphate buffer (50 mM, pH 7.4) containing 10% acetonitrile at 1.00 mL/min. The eluent was monitored by UV at 220, 254, and 280 nm. Under these conditions, the antibody eluted at ~ 8.4 min, and any aggregate material eluted at ~ 7.2 min. Excess small molecules eluted at approximately 11.3 min.

Hydrophobic Interaction Chromatography.

Hydrophobicity was evaluated through hydrophobic interaction chromatography (HIC) on a TSKgel Butyl-NPR column (14947) with an Agilent 1260 HPLC, where 20–50 μL samples were injected for analysis. Mobile Phase A: 25 mM Tris-HCL, 1.5 M ammonium sulfate pH 8.0. Mobile phase B: 25 mM Tris-HCl pH, 5% isopropanol. Linear gradient from 95% mobile phase A to 100% mobile B over 13 min with flow rate at 0.8 mL/min.

Plasma Stability Assessment.

Solutions of TROP2 ADCs (0.2 mg/mL) in 70% mouse and human serum and 30% PBS were prepared in sterilize conditions. The samples were kept in an incubator at 37 °C under CO2 gas. 50 μL aliquots were taken at day 0, 1, 4, and 7. Plasma proteins were crashed out of aliquots with 3 volumes of acetonitrile, and the supernatant was stored at −80 °C until analysis. Quantitative analysis of payload concentration was performed against a standard curve with liquid chromatography mass spectrometry (LCMS), using an MRM method. Samples were diluted with 2 volumes of water before injection.

Lysosomal Catabolism (Release of Exatecan, Figure S1).

Rat liver tritosomes (40 μL of 2.5 mg/mL, Xenotech) were activated by adding 40μL of 10 mM DTT/2 mM EDTA in NaOAc (0.2 M, pH 4.7) and heating the samples for 30 min at 37 °C. Each ADC (13 μg) was buffer exchanged into 0.2 M NaOAc (pH 4.7) using 30 kDa spin columns, concentrating to ~ 30 μL. The activated tritosomes (20 μL of ~ 1.25 mg/mL) were then added to each ADC and the mixture was heated to 37 °C. Aliquots (4 μL) were taken at 0, 5, 15, 30, 60, 120, and 240 min. The aliquots were immediately added to 35 μL of ACN to quench the enzymes, then centrifuged (10,000 rpm). The supernatant (~30 μL) was added to 75 μL of water and the samples were then frozen at −80 °C until the time of analysis. Analysis of released exatecan was performed via MRM/MS and daughter ions 375.29, 390.27, and 419.31 were used to quantify against an exatecan standard curve.

Lysosomal Catabolism (Release of Exatecan Derivatives, Figure 2).

Stock ADC solutions (78 μg in PBS) were buffer-exchanged into 0.2 M NaOAc buffer (pH 4.7) using a Vivaspin 500 centrifugal concentrator (10 kDa MWCO, Fisher Scientific, Cat. No. 14558396) giving postexchange concentrations ranging from 0.175 to 0.305 mg/mL. Mixed-gender IGS SD rat liver tritosomes (Avantor, Radnor, PA; Cat. No. NA3953002) were activated in 0.2 M NaOAc buffer (pH 4.7) by incubation with 10 mM dithiothreitol (DTT; Thermo Fisher Scientific) and 2 mM ethylenediaminetetraacetic acid (EDTA; VWR, Cat. No. BDH7314–1) at 37 °C for 30 min. The activation mixture (600 μL total) contained 240 μL tritosomes, 240 μL DTT, and 120 μL EDTA.

For the legumain-inhibited condition, 7.5 μL of 1 mM RR-11a (from a 10 mM DMSO stock; MedChemExpress, Monmouth Junction, NJ, USA; Cat. No. HY-112205) was added to 292.5 μL of activated tritosomes, gently mixed, and preincubated at 37 °C for 10 min prior to ADC addition. The final RR-11a concentration was 10 μM. For the no-inhibitor control, 7.5 μL DMSO was added instead under identical conditions. Subsequently, 20 μL of the tritosome mixture was combined with 30 μL of each ADC solution (total volume 50 μL) and incubated at 37 °C. All reactions were conducted in triplicate.

Aliquots (5 μL) were collected at 5 min, 0.5, 1, 2, 4, 6, and 24 h during incubation. Each aliquot was immediately quenched with 15 μL of ice-cold acetonitrile to terminate enzymatic activity and stored at −80 °C until analysis. Prior to LC–MS/MS analysis, samples were thawed on ice for 30 min, centrifuged at 13,200 rpm for 15 min at 4 °C, and a 15 μL portion of the clear supernatant was diluted with 45 μL of LC–MS grade water for injection. Quantitative analysis of released and conjugated payload species (Gly-Exa, βAla-Exa, GABA-Exa, Exa, and Dxd) was performed using a Xevo TQD triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with an electrospray ionization (ESI) source operating in positive ion mode. Data acquisition and processing were carried out using MassLynx and TargetLynx software (Waters Corp., Milford, MA, USA). Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm particle size; Waters Corp.) maintained at 40 °C. The mobile phase consisted of (A) water with 1% formic acid and (B) acetonitrile with 1% formic acid. The flow rate was 0.25 mL/min with the following gradient: 1% B (0–1.5 min), ramped to 80% B at 3.5 min, increased to 95% B at 4.0 min (held for 0.4 min), then returned to 1% B at 4.5 min and re-equilibrated until 6.0 min. The injection volume was 10 μL.

Mass spectrometric parameters were optimized as follows: source temperature, 200 °C; desolvation gas flow, 650 L/h; capillary voltage, 3.8 kV; cone voltage, 58 V; and collision energy, 3 V. The monitored multiple reaction monitoring (MRM) transitions (m/z) were: Gly-Exa, 493.32 → 419.25; βAla-Exa, 507.44 → 419.23; GABA-Exa, 521.44 → 86.14 and 419.23; Exa, 436.43 → 419.23; and Dxd, 494.40 → 450.03. Quantified concentrations of each released species (Gly-Exa, βAla-Exa, GABA-Exa, Exa, and DXd) were used to generate time-dependent release profiles. Nonlinear regression analysis (one-phase association model) was performed using GraphPad Prism (version 10.2; GraphPad Software, San Diego, CA, USA) to determine the rate and extent of payload release in the presence and absence of RR-11a.

Cytotoxicity of α-TROP2 Exatecan ADCs.

Cells were resuspended to 0.1 × 10^6 cells/ml in appropriate culture media supplemented with 10% FBS and 1x Pen Strep. 90ul of cell suspension were added to each well to provide a final cell density of 9000cells/well after treatment. ADCs were serial diluted at 10x the intended final concentration (30ug/mL to 0.0046ug/mL). A 10ul aliquot of each ADCs was added to the appropriate wells and the plate was incubated at 37C/5%CO2 for 6 days. Cell viability was assessed using the CellTiter-Glo assay kit (Promega).

Cytotoxicity of α-HER2 Exatecan ADCs in SKBR3 (HER2+) Cells.

SKBR3 cells were maintained in RPMI-1640 media supplemented with 10% FBS, with 10% Pen/Strep added. 90 μL of cells were seeded in a 96 well plate at a concentration of 0.1 million cells/mL, approximately 9,000 cells/well. A series dilution of each ADC was performed resulting in stock solutions at 10x the final test concentrations. The cells were allowed to adhere to the plate for 30 min, then 10 μL of ADC serial dilutions were added to the respective wells and the plate was left to incubate for 144 h (6 days). After the 144 h incubation period, the plate was analyzed using an XTT viability kit. 45 μL of activated XTT solution was added to each well then left to incubate for 4 h. At the end of the incubation period, the plate was read at 450 and 630 nm. Data was plotted using Graphpad Prism 9.0 to determine the IC50 values.

Bystander Killing Assay.

For the coculture-based bystander experiment, 9000 cells comprising of TROP2-positive BxPC-3 transduced to expressed green fluorescence protein (GFP) and TROP2-null AsPC-1 cells transduced to expressed red fluorescence protein(RFP) at ratio 4:1, were treated with ADCs at concentrations 3.3, 10, and 30 μg/mL and incubated for 6 days. Cellcyte X(Cytena) was used for live imaging of cells during the 6 days of incubation. After 6 days, end point images were collected using cellcyte X. Cells were harvested using Accutase(25–058Cl, Corning) and stained with TROP2 mAb labeled with sulfo-Cy5 dye. TROP2-positive and -null cells were analyzed by flow cytometry on a BD Accuri 6 flow cytometer (BD Biosciences). TROP2-positive and -null cells populations were determined using TROP2 and GFP expression.

DNA Damage Assessment Assay.

To assess for DNA damage induced my exatecan ADCs, 30000 BxPC-3 cells were seeded in 96 well plates and treated with ADCs with concentrations 10, 1 and 0.1ug/mL. This was incubated for 72hours. Subsequently, cells were detached using Accutase followed by stained with labeled antibodies specific for DNA damage markers; Anti-Hu PARP1(PE, eBioscience, REF:12–6668–42), Anti-H2A.X phospho(Ser139)(AF 647, Biolegend, Cat:613408) and Rabbit Anti-Active Caspase-3(FITC, BD biosciences, Cat:51–68654X). Cells were analyzed analyzed by flow cytometry on a BD Accuri 6 flow cytometer (BD Biosciences).¹⁰

Mouse Xenograft Study.

Human pancreatic cancer cells (CFPAC-1, ATCC) were cultured in IMDM(ATCC) media supplemented with 10% FBS and 1% Pen/strep, and maintained in exponential growth under the manufacturer’s recommended densities. 100 μL containing ~ 7.5 × 10⁶ cells suspension was implanted subcutaneously into the right flank of 8 weeks old male and female nude (Nu/J) mice (Jackson Laboratories). Tumor volume was recorded every 3 days using a caliper and estimated using the following formula: length × width²/2. ADC treatment was initiated once the tumor volumes reached 75–150 mm³. Mice were randomly assigned to single treatment groups (8 mice per group consisting of 4 males and 4 females). The mice were administered single dose with ADC (3 or 10 mg kg⁻¹), naked α-TROP2 mAb (10 mg kg⁻¹), isotype (10 mg kg⁻¹) or DPBS via intraperitoneal injection. The study was performed in a single-blind manner where the personnel treating the mice and measuring the tumors were unaware of the treatment group. Tumor volumes and body weights were measured every 3 days. For the large tumor study, tumors were allowed to grow to 400–800 mm³ volume before administering ADCs (3 mg kg⁻¹) intraperitoneally. Tumor measurement was conducted as described above.

Pharmacokinetics in Mice and Rats.

ADCs were administered intraperitoneally to (Nu/J) mice comprising of 2 male and 2 females per group at 3 mg kg⁻¹. Blood samples were obtained at 15 min, 6 h, 24 h, 48 h, 96 h, 7 days and 14 days after administration via submandibular vein or facial vein. Blood samples collected were centrifuged to give plasma samples which were kept on ice during processing and frozen at – 80 °C until analysis. Total mAb was determined using a Pixi automated ELISA (Correlia) following slight modifications of the manufacturer instructions. Standard curves for the ADCs (12.21 to 200,000 ng/mL) were generated using Swiss mouse serum (IMSNSASER100 ML, Innovative Research Inc.). Samples and standard concentrations prepared in serum were diluted 10-fold in DPBS. Samples and standard curves were prepared for analysis by a 10X dilution in Correlia sample diluent (part#: SD-010). An antihuman IgG assay cartridge was prepared for use according to manufacturer instructions. Detection antibody for human IgG (goat antihuman AF647, #PRI-2IGG) was diluted 25x and allocated into an 8 strip of PCR tubes. Samples were analyzed using the Correlia PIXI Electrophoresis module connected to the Opentron liquid handler.

Twelve male Sprague–Dawley rats (11–12 weeks old) were obtained from Charles River Laboratories. The animals were acclimatized for 1 week prior to the study and maintained on a 12h light/dark cycle with ad libitum access to standard chow and water. All animal procedures were conducted in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols (#868–21 for rats) from the Laboratory Animal Resources (LAR) at Binghamton University. Antibody-drug conjugates (ADCs) were administered intraperitoneally at a dose of 2.5 mg/kg. Blood samples were collected from the tail vein at multiple time points postdosing: 15 min, and 6, 24, 48, 96, 168, and 336 h. Plasma was separated by centrifugation at 4,500 rpm for 8 min and stored at – 80 °C until further analysis. The concentration of human IgG1 in rat plasma was determined using an Meso Scale Discovery (MSD) assay, following the manufacturer’s protocol (Meso Scale Discovery, 2024). Briefly, rabbit antihuman IgG (Thermo Fisher Scientific, Waltham, MA, USA; Catalog #31143) was used as the capture antibody on an MSD 96-well 1-spot plate. For detection, two sulfo-tagged antibodies were used: goat antihuman IgG (Meso Scale Discovery, Rockville, MD, USA; Catalog #R32AJ-5) and a mouse anti-Dxd monoclonal antibody (NovoPro, Shanghai, China; Catalog #176730) conjugated with MSD Gold sulfo-tag NHS-ester (Meso Scale Discovery, Catalog #R91AO-1). Plates were analyzed using the MESO QuickPlex SQ 120MM system (Meso Scale Discovery). Calibration standards ranging from 3.9 ng/mL to 1,000 ng/mL of antibody-drug conjugates (ADCs) were used to quantify total humanized IgG1 concentrations. The concentration–time data were analyzed by noncompartment pharmacokinetic analysis using Phoenix WinNonlin Version 8.4 (Certara, NJ, USA)

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c02681.

Supporting figures (S1–S13) and tables (S1 and S2) (PDF)

Molecular strings and biological data for all small molecule compounds (CSV)

ABBREVIATIONS

ADC

antibody-drug conjugates

AHX

6-aminohexanoic acid

DAR

drug-to-antibody ratio

DMA

dimethylacetamide

Exa

exatecan

GABA

gamma-aminobutyric acid

HIC

hydrophobic interaction chromatography

mc

maleimide caproyl

MDR

multidrug resistance

MMAE

monomethyl auristatin E

MMAF

monomethyl auristatin F

mp

maleimide propionyl

PABC

para-amino benzyl carbamate

PDAC

pancreatic ductal adenocarcinoma

PARP1

poly(ADP-ribose) polymerase 1

SD rats

Sprague–Dawley rats

SEC

size exclusion chromatography

SIR

single ion resonance

TCEP

tris(2-carboxyethyl)-phosphine

TNBC

triple negative breast cancer

Top1i

topoisomerase I inhibitor

vcMMAE

mcValCitPABC-MMAE